The research proposed for the second year shall focus on the amino acid sequence of the small BrCN-derived hemepeptides of P-450CAM, P-450LM-2, P-450LM-4, P-450scc and P-45011 beta. The objective is to apply a microsequencing technique making use of solid phase Edman degradation in combination with highly selective enzymatic cleavages to generate peptides of intermediate size which can be sequenced without further derivatization by field desorption mass spectrometry. The hemepeptides will also be digested by the post proline cleavage enzyme and the resulting fragments will be further degraded by cathepsin C. The dipeptides released by this enzyme will be identified by conventional mass spectrometry. This approach is best suited for unequivocal determination of photocovalently attached substrate and inhibitor derivatives, the nature of the contact residue(s) and the exact C-atom(s) within the residue(s). Thus comparative sequencing in conjunction with photoaffinity labeling using a variety of arylazido derivatives as labels for substrate and inhibitor binding sites shall reveal the exact mode of binding of substrate and inhibitor molecules to P-450 hemeproteins. It is hoped that this information will explain the difference between P-450 hemeproteins of narrow and of broad substrate specificities and reveal common structural features of their heme environment. From structural permutation of the labels we hope to learn more about the distance between sustrates and the heme group as well as to get a glimpse at the geometry of the substrate binding site. Immunochemical experiments will be pursued with antibodies against the 5 hemeproteins listed above and their hemepeptides, and any other purified P-450 which might become available in the meantime.